1. Technical Field
This invention pertains generally to one-step sample preparation and analysis, and more particularly to the integration of suspension separation, multiplexed compartmentalization and digital amplification and/or detection of components within the separated solution, where the system may be automated using degas driven flow.
2. Background Discussion
Real-time PCR is currently the standard method for quantitative nucleic acid (NA) detection in body fluid samples. Viral load, or the quantity of virus in an organism (usually blood), is one of the most essential markers for indicating the effectiveness of antiviral therapy and disease progression. Conventional HIV viral load monitoring tests, approved by the US Food and Drug Administration, use real-time polymerase chain reaction (real-time PCR) assays. This method typically involves expensive equipment, such as real-time thermal cyclers, 2-3 hours of assay time, multiple manual steps requiring trained technicians, and the need for sample preparation to remove contaminants. For example, in standard real-time PCR assays, a sample such as blood needs to be purified since hemoglobin and IgG can inhibit polymerase activity as their chelating nature disrupts Fe3+ concentration.
Since blood cells can interfere with diagnostic assays by obstructing the optical detection path by its opaqueness, plasma separation is a common step for blood based protein and other sample component diagnostics. Hemoglobin released from lysed red blood cells can inhibit other enzyme reactions by chelating ions and therefore it is desirable to remove blood cells prior to conducting almost all assays.
Sample purification may be done with phenol/chloroform extraction or silica spin columns. The standard plasma separation technique is via centrifugation, which requires electrical sources and bulky equipment. Membrane filter and mechanical filter methods are also popular; however, they often clog or cause hemolysis. Other methods that utilize hydrodynamic lift force, Zweifach-Fung effect or inertia forces require external pumps to control flow rates precisely. Active separation using external fields such as acoustics, electro-osmotic flow, and magnetic forces have been used. However, these separations also require external power sources, have highly complex chip design, and require external equipment.
There are also sedimentation methods, such as cross flow-filtration, sedimentation in a plug and gravity induced lamination. The main advantage of these sedimentation systems is the significant reduction in hemolysis because of the low shear stress on red blood cells. However, of all the discussed purification or separation methods, there has yet to be a coupling of these technologies and sample compartmentalization for a rapid one-step digital fluid sample analysis.
Other NA assays, such as transcription-mediated amplification or branched-DNA tests, can be used but suffer from the same constraints as real-time PCR, requiring multiple steps of sample preparation, approximately 3 to 6 hours of assay time and highly trained technicians. Furthermore, these techniques all require centralized laboratory testing and, therefore, samples have to be transported, which can result in sample degradation. Centralization also limits the access for low resource sites that are far away.
Newer ELISA (Enzyme Linked Immunosorbent Assay) based techniques have also been developed. Although they can reduce the cost of testing (approximately $5 to $23) and are simpler assays to perform, they are still time consuming, requiring significant manual handling time (6 to 72 hours). The latest lateral flow strips have been shown to detect NA. However, multiple manual steps are still required and these assays generally provide qualitative but not quantitative NA detection.
It is desirable to combine rapid sample preparation and quantitative assay endpoint readout into the same diagnostic chip to simplify, reduce the cost and shorten the steps needed for fluid sample analysis.